Single-Cell Transcriptional Archetypes of Airway Inflammation in Cystic Fibrosis

Abstract

Rationale: Cystic fibrosis (CF) is a life-shortening, multisystem hereditary disease caused by abnormal chloride transport. CF lung disease is driven by innate immune dysfunction and exaggerated inflammatory responses that contribute to tissue injury. To define the transcriptional profile of this airway immune dysfunction, we performed the first single-cell transcriptome characterization of CF sputum.Objectives: To define the transcriptional profile of sputum cells and its implication in the pathogenesis of immune function and the development of CF lung disease.Methods: We performed single-cell RNA sequencing of sputum cells from nine subjects with CF and five healthy control subjects. We applied novel computational approaches to define expression-based cell function and maturity profiles, herein called transcriptional archetypes.Measurements and Main Results: The airway immune cell repertoire shifted from alveolar macrophages in healthy control subjects to a predominance of recruited monocytes and neutrophils in CF. Recruited lung mononuclear phagocytes were abundant in CF and were separated into the following three archetypes: activated monocytes, monocyte-derived macrophages, and heat shock-activated monocytes. Neutrophils were the most prevalent in CF, with a dominant immature proinflammatory archetype. Although CF monocytes exhibited proinflammatory features, both monocytes and neutrophils showed transcriptional evidence of abnormal phagocytic and cell-survival programs.Conclusions: Our findings offer an opportunity to understand subject-specific immune dysfunction and its contribution to divergent clinical courses in CF. As we progress toward personalized applications of therapeutic and genomic developments, we hope this inflammation-profiling approach will enable further discoveries that change the natural history of CF lung disease.

Keywords: RNA-seq; cystic fibrosis; macrophages; monocytes; neutrophils.

Figure 1.

Single-cell RNA sequencing reveals an immune cell repertoire shift from alveolar macrophages to recruited monocytes and polymorphonuclear neutrophils in cystic fibrosis (CF). (A) Schematic of the experimental design. Spontaneously expectorated sputum from patients with CF and induced sputum from healthy control subjects was collected. Sputum was processed into a single-cell suspension. Droplet-based single-cell RNA sequencing barcoding, library preparation, sequencing, and computational analysis were performed. (B) Uniform manifold approximation and projection visualization of 20,095 sputum cells from nine patients with CF and five control subjects. Each dot represents a single cell, and cells are labeled by 1) cell type, 2) disease state, and 3) subject. (C) Heatmap of marker genes for all cell types identified. Each column represents the average expression value of one subject grouped by disease status and cell type. Gene expression values are unity-normalized from 0 to 1. (D) Boxplots showing percentages of all identified cell types to all cells profiled per subject, separated by disease state. Whiskers represent 1.5× interquartile range. *P < 0.05 determined by a Wilcoxon rank sum test comparing cell percentages of patients with CF and control subjects. alvMΦ = alveolar macrophage; B = B-lymphocyte; cDC = classical dendritic cell; HC = healthy control; Mo = monocyte; MoMΦ = monocyte-derived macrophage; pDC = plasmacytoid dendritic cell; PMN = polymorphonuclear neutrophil; T and NK = T-lymphocytes and NK cells.

Figure 2.

Recruited lung mononuclear phagocytes are a distinct cell population with a broad spectrum of maturity and immune activation in cystic fibrosis airways. (A) Potential of heat diffusion for affinity-based transition embedding (PHATE) of monocytes and monocyte-derived macrophages colored by pseudotime, all starting from quiescent monocytes toward 1) activated monocytes, 2) mature monocyte-derived macrophages, 3) monocytes expressing a heat-shock response, and 4) monocytes and monocyte-derived macrophages, colored by disease state. All three archetypes are accompanied by three PHATE plots colored by the gene expression of typical genes, ramping up along a specific pseudotime. For the corresponding uniform manifold approximation and projection embedding colored by the gene expressions of the same genes, see Figure E4. For the corresponding PHATE colored by cell type and subjects, see Figure E5. (B) Heatmap of gene expression and regulon activity in monocytes undergoing activation (ordered by pseudotime distances along PHATE manifolds that transition from quiescent monocytes toward an activated monocyte archetype). (C) Heatmap of gene expression and regulon activity in monocytes undergoing maturation (ordered by pseudotime distances along PHATE manifolds that transition from quiescent monocytes toward a control-enriched mature monocyte-derived macrophage archetype). In both heatmaps, annotation bars represent the pseudotime distance, disease status, and subject for each cell; expression values are centered and scaled. (D) Violin plots of pathway activity scores grouped by cell type and separated by disease state. Depicted pathway scores from left to right are as follows: GO:0045087 innate immune response, GO:0006958 complement activation and classical pathway, GO:0019882 antigen processing and presentation, and GO:0006911 phagocytosis and engulfment. *False discovery rate–adjusted P values < 0.05 calculated using the Wilcoxon signed-rank test. alvMΦ = alveolar macrophage; CF = cystic fibrosis; Gene Expr. = gene expression; HC = healthy control; Mo = monocyte; MoMΦ = monocyte-derived macrophage; PMN = polymorphonuclear neutrophil; Regulon Act. = regulon activity.

Figure 3.

An immature proinflammatory archetype prevails among cystic fibrosis (CF) airway polymorphonuclear neutrophils (PMNs). (A) Potential of heat diffusion for affinity-based transition embedding (PHATE) of PMNs colored by 1) pseudotime from immature to mature PMNs, 2) examples of canonical marker features of immaturity (CXCR4) and maturity (FCGR3B and CXCR2) in peripheral PMNs, and 3) disease state. The cells deviating upward are PMNs expressing heat-shock response genes. For PHATE colored by gene expression of HSPA1A, HSPH1, and DNAJB1, see Figure E6A. For corresponding PHATE colored by disease state and subjects, see Figure E6B. (B) Heatmap of gene expression and regulon activity in PMNs ordered by pseudotime distances along PHATE manifolds that transition from CF-enriched regions of the immature and activated PMN archetype toward control-enriched mature PMN archetype. Annotation bars represent the pseudotime distance, disease status, and subject for each cell; expression values are centered and scaled. (C) Violin plots of differentially expressed genes comparing CF and control PMN populations (for P values, see Data file E3) grouped by disease state and sorted thematically. HC = healthy control; MHC = major histocompatibility complex.